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siPORT™ NeoFX™ Transfection Agent
siRNA Transfection Agents:
Not All are Created Equal
Gene silencing experiments call for critical, but sometimes time-consuming optimization steps to maximize transfection efficiency and maintain cell viability. Careful optimization involves selecting the most appropriate siRNA delivery method and transfection agent. However, once a protocol has been optimized for a particular cell type, reproducible siRNA screening experiments can be easily performed. This article shows the wide variation in the efficacy of different transfection agents in two cell lines and underlines the importance of appropriate transfection agent selection.
Selecting the Appropriate Transfection Agent
There are numerous transfection reagents available to researchers interested in gene silencing using siRNAs. The efficacy of these transfection agents and the conditions used for transfection are cell type specific and vary widely, affecting knockdown of gene expression and cell viability. To achieve maximum gene knockdown while maintaining an acceptable level of viability for the cell type under study, one must test for the most appropriate transfection agent and transfection conditions.
Robust siRNA Transfection in Just 24 Hours
Ambion has been developing products to support RNAi research for over 5 years. The siPORT™ NeoFX™ Transfection Agent was designed to refine siRNA transfection protocols resulting in decreased optimization time. This lipid-based formulation can be used very effectively with standard transfection protocols, and the reagent is particularly useful for efficient reverse transfection of adherent cells—as they are subcultured—without increased cytotoxicity. For reverse transfection, siPORT NeoFX is added to the diluted siRNA, incubated to form transfection complexes, the complexes are added to culture dish wells, and overlayed with cells. From start to finish, successful gene silencing experiments can be completed in as little as 24 hours.
siPORT NeoFX vs. the Competition
In Figure 1, siPORT NeoFX Transfection Agent was compared to transfection agents from other leading manufacturers for the reverse transfection of siRNA targeting GAPDH into two cell lines: HeLa (human adenocarcinoma) and HepG2 cells (human hepatocellular carcinoma; a cell line which has been traditionally challenging to transfect). Following reverse transfection, cells were lysed with KDalert™ Lysis Buffer, and an aliquot from each well was assayed for GAPDH enzymatic activity. The data indicate that siPORT NeoFx Transfection Agent is a highly robust reagent across a range of concentrations in both HeLa and HepG2 cells (Panels A and B, respectively). siPORT NeoFx Transfection Agent also exhibits minimal toxicity, compared to the other reagents, as determined by the KDalert™ GAPDH Assay Kit. Other data confirm that siPORT NeoFx does not significantly affect viability in a wide range of cell types over a range of reagent volumes, whereas the other reagents show a much stronger cytotoxic response when used at similar concentrations.
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Figure 1. siPORT™ NeoFX™ Transfection Agent Efficiently Transfects Cells with Low Cytotoxic Effects. HeLa (A) and HepG2 (B) cells were reverse transfected [6 X 103 cells per well in four 96 well plates (Silencer® CellReady™ Transfection Optimization Plates) containing GAPDH siRNA or Negative Control #1 siRNA] in duplicate using three transfection agents (siPORT NeoFX, Reagent Q, and Reagent D1) at the indicated volumes. Following reverse transfection, cells were grown at 37°C for a total of 72 hr with a culture medium change at 24 hr post transfection. Cells were lysed with KDalert™ Lysis Buffer, and an aliquot from each well was assayed for GAPDH enzyme activity. Cell viability is represented as a percentage of GAPDH enzyme activity of negative control siRNA transfected cells versus untreated cells. Remaining gene expression is represented as the percentage of GAPDH mRNA remaining in GAPDH siRNA treated cells compared to negative control treated cells. |
Additional Benefits
In addition, the transfection agent is stable at a range of temperatures, and can even be frozen and thawed prior to use without affecting activity. The transfection complexes are active and stable even in the presence of serum, so for some cell types, there is no need to remove or replace the culture medium following transfection.
What You Get
siPORT NeoFX Transfection Agent is available in sizes of 0.4 ml and 1.0 ml, which is enough to perform up to 800 and 2000 transfections, respectively, in 96 well tissue culture plates. Instructions for transfection optimization are provided in the detailed protocol accompanying the transfection agent. This versatile transfection agent is also part of Ambion optimization and starter kits (see sidebar).
Scientific Contributor
Anton Zimmerman • Ambion, Inc.
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